Dysregulated monocyte sequestration and angiogenesis clearly play a deleterious role in the joints of rheumatoid arthritis (RA) patients. The CX3C chemokine, fractalkine (fkn), is structurally unique and functionally versatile. In conjunction with its receptor, CX3CR1, fkn can act directly as both an adhesion molecule and a chemoattractant for monocytes, thus suggesting it may contribute to the sustained inflammatory response occurring in the RA synovium. Data presented here suggest that )1 the soluble form of fkn (sfkn) is sufficient to induce endothelial cell (EC) chemotaxis, EC tube formation on Matrigel in vitro and angiogenesis in vivo consistent with a pro-angiogenic role for sfkn; 2) CX3CR1 s expressed by endothelial cells; 3) the fkn present in RA, synovial tissue is capable of inducing angiogenesis; and 4) the sfkn in RA synovial fluid is capable of inducing monocyte migration and angiogenesis. Therefore, we propose to examine the role of the seven transmembrane CX3CR1 receptor as well as the G proteins with which it associates. Our hypothesis is: (1) fkn- and sfkn-induced adhesion, chemoattraction, and angiogenesis in RA each occur via proteins in monocytes or ECs will inhibit fkn-associated functions induced by RA synovial fluid and synovial tissue. Through the use of overexpressed dominant-negative peptides that can inhibit the association of G proteins with their cognate receptors, we will accomplish three of our specific aims, which are to delineate which G proteins are responsible for mediating sfkn- and RA synovial fluid-induced monocyte chemotaxis, monocyte/ endothelial cell adhesion, and angiogenesis. As a first step towards identifying novel inhibitors of CX3CR1 and potential new therapies, our fourth specific aim will then utilize the above information to identify short peptide sequences that can specifically inhibit CX3CR1 from associating with G proteins. Finally, we will use these new inhibitors to determine the impact of uncoupling CX3CR1 from its associated G proteins in chemotaxis, adhesion, and angiogenesis assays induced by sfkn and RA SF. These experiments should provide extensive new insights about the mechanism of fkn signaling through CX3CR1 and its relationship to cellular function.